The goal of this project is to develop and clinically test a unique direct-acting thrombolytic enzyme, plasmin that has the potential to provide safe thrombolytic therapy. Intracranial hemorrhage complicates thrombolytic therapy using plasminogen activators in up to 2 percent of patients with peripheral arterial occlusion and 10 percent of patients with ischemic stroke. Our approach will focus on the biochemical properties of plasmin which provide heretofore unutilized advantages over currently-approved thrombolytic agents, which are indirect-acting plasminogen activators. Our prior studies show that plasmin delivered locally in vitro or in vivo is as effective as TPA for thrombolysis, even more effective when local plasminogen content is limited. Furthermore, animal studies show that plasmin is safer than TPA, devoid of hemorrhagic consequence even at 4-fold higher doses than needed for thrombolysis. The proposed studies are designed to translate our fundamental observations to clinical practice. Specific aim 1) is to evaluate the hemostatic safety of plasmin in animal models of bleeding, using the rabbit ear puncture model to evaluate antiplasmin as a safety monitoring tool and as an antidote for bleeding and a rabbit embolic stroke model to assess the risk for parenchymal (intracranial) hemorrhage. Specific aim 2) is to translate basic observations to Phase I studies in humans, specifically, for catheter delivery to adult and pediatric patients with thrombosed venous access catheters, for local intravenous treatment of upper extremity deep vein thrombosis, and for intra-arterial treatment of acute ischemic stroke. The animal studies will be performed at Los Angeles Orthopedic Hospital/UCLA, and the clinical trials will take place in the Center for Health Sciences/David Geffen School of Medicine at UCLA. We hypothesize and anticipate that plasmin will provide clinically effective thrombolysis without an increased risk of bleeding .